The term "antigen" is defined as anything that can serve as a target for an immune response. The immune response can be either cellular or humoral. The term "vaccine" is defined herein as a suspension or solution of antigenic moieties, usually consisting of infectious agents, or some part of the infectious agents, that is injected into the body to produce active immunity. The antigenic moiety making up the vaccine can be either a microorganism or a natural product purified from a microorganism, a synthetic product or a genetically engineered protein, peptide, polysaccharide or similar product. The term "cell mediated immunity" is defined as an immune response mediated by cells rather than by antibody. It includes, but is not limited to, delayed type hypersensitivity and cytotoxic T cells. The term "adjuvant" as used herein is any substance whose admixture with an injected immunogen increases or otherwise modifies the immune response. A "hapten" is defined herein as a substance that reacts selectively with appropriate antibodies or T cells but the hapten itself is usually not immunogenic. Most haptens are small molecules or small parts of large molecules, but some macromolecules can also function as haptens. The term "conjugation" is defined herein as the covalent or other form of linking two or more molecules. It can be accomplished either by chemical means or in vivo by biologic means such as genetic engineering. The term "isotype" is a subtype of an antibody. The term "lipopolysaccharide" (LPS) is a amphipathic glycophospholipid obtained from the outer membrane of gram-negative bacteria which has a hydrophobic moiety called lipid A and a sugar moiety (polysaccharide or oligosaccharide). The term "non-toxic LPS" is defined as an LPS with very low toxicity based on one or more measurements of 50% lethal dose in animals (LD.sub.50), 50% chick embryo lethal dose (CELD.sub.50), pyrogenicity in rabbit, or dermal Shwartzman reaction. The in vitro measurements of the induction of either/both tissue necrosis factor and IL-1 by macrophage can also be used to determine the toxicity of LPS. The term "detoxified LPS" is defined as being LPS with reduced toxicity due to chemical modification of the structure of Lipid A moiety, i.e., removal of one phosphate group, removal of one to three fatty acyl groups, the introduction of new functional groups (e.g., methyl, acetyl, alcohol and the like), or partial reduction or oxidation.
An effective vaccine must induce an appropriate response to the correct antigen or antigens. There are several distinct types of immune responses which vary in their ability to confer protection against particular diseases. For example, antibodies may confer protection against bacterial infections, but cell mediated immunity is required for eliminating from the body many viral infections and tumors. There are multiple distinct types of antibody and cell-mediated immune responses. Cell-mediated responses are divided into two basic groups: 1) delayed-type hypersensitivity in which T cells act indirectly via macrophages and other cells or cell products, and 2) cytotoxicity in which specialized T-cells specifically and directly attack and kill infected cells.
There are five major classes of antibody: IgM, IgG, IgE, IgA and IgD. These classes have distinct functions in the immune response. IgG, the dominant class in the blood, is subdivided into several different subclasses or isotypes. In mice, these isotypes are IgG1, IgG2a, IgG2b, and IgG3. In humans, the isotypes are IgG1, IgG2, IgG3 and IgG4..sup.1 Similar isotypes have been defined in most other mammalian species in which they have been investigated. The nomenclature of IgG isotypes is different in different species because the names were coined before the structure or function of the antibody isotypes were understood. Although much still remains to be learned, the IgG isotypes appear to be highly conserved among mammalian species. FNT .sup.1 Clark, W. R., The Experimental Foundations of Modern Immunology, Chapter 4, "The properties and fine structure of immunoglobulins", pgs. 62-74, John Wiley & Sons
The IgG isotypes differ in their ability to confer protection to particular infections. IgG2a and IgG2b in mice activate complement, mediate antibody mediated cell mediated cytotoxicity and other functions. They are particularly effective in conferring protection against many bacterial, viral and parasitic infections. The counterparts in humans appear to be IgG1 and IgG3. In contrast, murine IgG3 is particularly effective in conferring protection against bacteria with polysaccharide coats such as the pneumococcus. The human counterpart seems to be IgG4. Isotypes such as IgG1 in mice do not fix complement, neutralize toxins effectively, but are markedly less effective for many bacterial and viral infections. Because the different IgG isotypes differ markedly in their ability to confer immunity, it is important that vaccines induce the most appropriate isotype for a particular infection. Even though the nomenclature is different, available evidence and modem theory indicate that the properties of immunogens which determine the isotype of antibody produced are similar across mammalian species. In other words, an immunogen which stimulates delayed type hypersensitivity or complement fixing IgG antibody in one species will generally stimulate similar responses in other species.
Biosynthetic and recombinant DNA technology is permitting development of vaccines possessing antigenic epitopes that were previously impossible to produce. Current vaccine candidates include virtually all infectious agents, allergens and even host components such as hormones and molecules involved in autoimmune diseases, cancer and other diseases. The infections agents include, but are not limited to, viruses, bacteria, parasites, rickettsiae and fungi. Hormones are being evaluated as vaccines for diverse purposes such as prevention of pregnancy and treatment of disease. Vaccines for treatment of cancers, such as melanoma, are being evaluated in animals and man. In each case, optimal effect of the vaccine depends upon stimulating the appropriate type, intensity and duration of the immune response.
The work on the parasitic disease malaria is especially important. This disease affects in excess of 200 million people per year worldwide and is the most important disease in the world in terms of morbidity and loss of work. The techniques of genetic engineering have been used to identify, and now to produce in substantial quantities, several peptides and proteins associated with malarial parasites. In particular, a twelve amino acid peptide from the sporozoite stage has been determined to carry an important antigenic site. Antibodies against this particular peptide can kill the parasite immediately after it is injected. Unfortunately, this peptide, by itself, does not produce an adequate immune response. Each species of malaria has a different peptide, but the characteristic structure and repeat units is found in all of them.
In an effort to induce an effective immune response to the sporozoite peptide, the peptide has been conjugated with carriers and administered with adjuvants. To date, however, the adjuvants used with the peptide or peptide conjugates have not produced satisfactory results. Similarly important antigens have been identified on the blood stages of malarial parasites, but available vaccine formulations have been unable to induce protective immunity.
Human immunodeficiency virus (HIV) causes AIDS. Many recombinant and peptide antigens have been prepared from HIV. There is evidence that antibodies against these antigens can neutralize the virus and that the body's immune response is able to prevent or control infections. However, generally effective vaccines to induce protective immune responses against HIV have remained an elusive goal. Hemophilus influenza and Pneumococcal pneumonia provide further examples. The important antigens of these bacteria are polysaccharides which elicit protective immune responses poorly in infants and elderly persons who are in most danger from these infections. Similar situations exist for numerous other viral, bacterial and parasitic infections in addition to tumors and other diseases which can be modulated by immune responses. Modem science has provided the means to identify and produce antigens from most conditions which are influenced by immune responses. The failure of many new antigens to induce optimal protection has highlighted an increasing need for means to influence the type, intensity, and duration of immune response produced by vaccines.
Thus, interest has arisen in the development of potent, nontoxic adjuvants that will enhance, and perhaps more importantly, modulate the immunogenicity of haptenic epitopes. In addition, adjuvants are-needed for use with conventional vaccines to elicit an earlier, more potent, or more prolonged response of the appropriate type. Such an adjuvant would also be useful in cases where antigen supply is limited or is costly to produce.
The development of adjuvants has, until recently, been empirical. An enormous number of compounds have been found to modulate the immune response. These compounds have been notably diverse in both substance and function, a fact that has complicated attempts to discover the unifying mechanisms of adjuvant action. The elucidation of these mechanisms has lagged behind recent advances in the understanding of the immune system.
This diversity of adjuvants has presented difficulties in their classification. Adjuvants are occasionally grouped according to their origin, be it mineral, bacterial, plant, synthetic, or host product. The first group under this classification are the mineral adjuvants, such as aluminum compounds. The first use of aluminum compounds as adjuvants was described in 1926. Since that time antigens precipitated with aluminum salts or antigens mixed with or adsorbed to performed aluminum compounds have been used extensively to augment immune responses in animals and humans. Aluminum compounds and similar adjuvants appear to work through the following mechanism. The aluminum physically binds to the antigen to form particles. These form a depot of antigen in tissue following injection. Excretion of the antigen is slowed, thus prolonging the time of interaction between the antigen and antigen-presenting cells such as macrophages or follicular-dendritic cells. In addition, immunocompetent cells are attracted to the area of injection and are activated. Aluminum particles have been demonstrated in regional lymph nodes of rabbits seven days following immunization, and it may be that another significant function is to direct antigen to T cell containing areas in the nodes themselves. Adjuvant potency has been shown to correlate with intimation of the draining lymph nodes. While many studies have confirmed that antigens administered with aluminum salts led to increased humoral immunity, cell mediated immunity appears to be only slightly increased, as measured by delayed-type hypersensitivity. Aluminum hydroxide has also been described as activating the complement pathway. This mechanism may play a role in the local inflammatory response as well as immunoglobulin production and B cell memory.
Primarily because of their excellent record of safety, aluminum compounds are presently the only adjuvants used in humans. They are, however, not without problems. Aluminum containing vaccines occasionally cause local reactions. Although allergic manifestations are not usually a clinical problem, aluminum compounds have been also said to attract eosinophils to the area of injection via a T-cell-dependent mechanism, to induce an IgE response if injected after antigen priming, and to elicit a carrier-specific cell population with helper function for IgE response. In addition, aluminum-containing vaccines cannot be lyophilized, thus necessitating refrigerated transport and storage with the resulting risk of contamination.
Finally, and most importantly, aluminum compounds are not always successful in inducing sustained protection from disease. This is due, in part, to their inability to induce the most appropriate isotypes of antibody or the optimal type of cell-mediated immunity. Thus, while aluminum salts have been a sufficient adjuvant for strong immunogens that require only antibody responses to elicit protection, they are not effective when used with weak immunogens like synthetic peptides of malaria or for introducing cell-mediated immune responses or IgG isotype of the type required to fight infections.
Another large group of adjuvants are those of bacterial origin. Adjuvants with bacterial origins have recently been purified and synthesized (e.g. muramyl dipeptides, lipid A) and host mediators have been cloned (Interleukin 1 and 2), providing chemically characterized products for study. The last decade has brought significant progress in the chemical purification of three adjuvants of active components of bacterial origin: Bordetella pertussis, lipopolysaccharide and Freund's Complete Adjuvant (FCA).
B. pertussis is of interest due to its ability to modulate cell-mediated immunity through action on T-lymphocyte populations. For lipopolysaccharide and Freund's Complete Adjuvant, adjuvant active moieties have been identified and synthesized which permit study of structure-function relationships.
Lipopolysaccharide and its various derivatives, including lipid A, have been found to be powerful adjuvants in combination with liposomes or other lipid emulsions. It is not yet certain whether derivatives with sufficiently low toxicity for general use in humans can be produced. Freund's Complete Adjuvant is the standard in most experimental studies. However, it produces severe local and systemic inflammatory reactions which may be severe enough to cripple or kill the host. It cannot be used in humans and may be banned for use in animals.
Many other types of materials have been used at various times as adjuvants. They include plant products such as saponin, animal products such as chitin and numerous synthetic chemicals. The source of an adjuvant among these categories has not proved particularly useful in predicting its biological properties.
Adjuvants have also been categorized by their proposed mechanisms of action. This type of classification is necessarily somewhat arbitrary because most adjuvants appear to function by more than one mechanism. Adjuvants may act through antigen localization and delivery, or by direct effects on cells making up the immune system, such as macrophages and lymphocytes. Another mechanism by which adjuvants enhance the immune response is by creation of an antigen depot. This appears to contribute to the adjuvant activity of aluminum compounds, oil emulsions, liposomes, and synthetic polymers. The adjuvant activity of lipopolysaccharides and muramyl dipeptides appears to be mainly mediated through activation of the macrophage, whereas B. pertussis affects both macrophages and lymphocytes. Recent and speculative approaches to immunopotentiation, such as the utilization of monokines and lymphokines, and the manipulation of the antigen, carrier, and adjuvant to augment the immune response are currently fashionable.
Small immunogens, such as the synthetic peptide of malaria, can be attached to larger proteins or other carriers to increase the immune response. The relationship between molecular size and complexity of an antigen relative to immunogenicity reflects the availability of antigenic determinants on the molecule. This relationship was first noted by Landsteiner when he demonstrated the need to complex small radicals with larger (carrier) molecules to stimulate an immune response. However, the mechanistic basis for the requirement was to await experiments that demonstrated the carrier effect and the need for a minimum of two antigenic determinants on a molecule to express immunogenicity. These determinants represented the carrier and haptenic determinants that interact with T and B lymphocytes, respectively. However, the influence of the carrier moiety extends beyond simple antigenicity through activation of T cells in T-dependent humoral responses.
The combination of determinants on an antigen molecule can influence the immune response by differential activation of various types of helper and suppressor T cells. A model system demonstrating this effect is the genetically controlled humoral response of responder (C57B1/6) and non-responder (DBA/1) mice to the synthetic terpolymer 1-glutamic acid.sup.60 -L-alanine.sup.30 -L-tyrosine.sup.10 (GAT). While C57B1/6 mice respond to this polypeptide, DBA/1 mice will respond only if the GAT is coupled to methylated bovine serum albumin (MBSA). However, if the mice are injected with GAT prior to immunization with GAT-MSBA, a detectable antibody response to GAT does not occur. The explanation for these observations is that GAT stimulates helper T cells in the responder mice but preferentially activates suppressor T cells in non-responder mice. This predominance of suppressor cells prevents a response to GAT even when coupled to MBSA. However, if primary immunization is with GAT-MBSA, activation of helper T cells by the carrier moiety provides help that overrides the effect of any suppressor cells activated by GAT.
Determinants associated with a native protein molecule have also been demonstrated to contribute differently to help and suppression. Conjugation of an immunogenic carrier to an antigen can change the isotype of antibodies produced in response to that antigen. Purified polysaccharides from many encapsulated bacteria are thymus-independent antigens due to their polymeric nature with multiple repeating antigenic determinants. While they represent protective antigens of these bacteria, the IgM antibodies produced have limited efficacy in preventing disease. This is largely due to their inability to stimulate immunologic memory or adequate immune responses in very young or old individuals who are at high risk from the infections. Therefore, polysaccharides from Neisseria meningitidis and Haemophilus influenza type b have been conjugated to proteins, such as tetanus toxoid. These conjugated preparations act as thymus-dependent antigens and induce IgG responses to the polysaccharide moiety as well as immunologic memory. They also induce responses in young or old individuals. Likewise, the thymic-independent polysaccharide carriers have little potential for enhancing the immunogenicity of peptides, such as those involved with malaria which require thymic-dependent IgG immune responses.
Publications by Feldmann and Lee and others state that flagella antigens of Salmonella organisms are typical thymic-independent antigens which stimulate strong IgM antibody responses..sup.2,3 They stimulate only late maturing B cells which are absent from infants. Such immunogens also tend to induce tolerance in infants and do not induce memory or other aspects of the complex immune responses induced by thymic-dependent antigens in adults. This published data would lead one to believe that they have little potential as adjuvants or carriers for malaria peptides or other small antigens which require thymic-dependent IgG antibody responses. FNT .sup.2 Feldmann, M., et al., "The Relationship Between Antigenic Structure and the Requirement for Thymus-Derived Cells in the Immune Response", J. Exp. Med., Vol. 134, pgs. 103-119 (1971) FNT .sup.3 Lee, et al., "Decline and Spontaneous Recovery of the Monoclonal Response to Phosphorylcholine During Repeated Immunization", J. Immun., Vol. 113, pgs. 1644-1646 (1974)
There probably is no precise point of transition that distinguishes a carrier from an adjuvant. The carrier moiety is contributory to a property of antigens that has been termed intrinsic adjuvanticity. The capacity of certain materials to convert a tolerogen to an immunogen has been termed as extrinsic adjuvanticity. Adjuvanticity can be enhanced by increasing the size of the antigen through aggregation of proteins or adsorption to immunogenic or inert carriers. Thus materials, such as aluminum hydroxide, latex particles, bentonite, or liposomes that adsorb antigen and enhance the immune response, are termed adjuvants. However, this observed effect of aggregation of antigen represents only a limited view of adjuvant actions which are now recognized as being extremely complex.
Small peptides and other haptens are incapable of evoking a strong immune response without the use of an adjuvant. Most adjuvants that are currently available are toxic and/or do not evoke an immune response that is effective in protecting the animal or human against infection with the infectious agent. Thus, what is needed is a vaccine which can be administered to an animal or human and will cause the immune system to mount a prolonged and potent immune response of the correct type against an appropriate antigen.
Large hydrophobic nonionic block copolymer surfactants have been shown to be effective immunologic adjuvants which are potentially useful in man..sup.4,5,6 They appear to act as adhesives which bind protein antigens to the surface of oil drops and/or cells in a way which facilitates antigen presentation. Previous studies have demonstrated that these copolymers can induce high liter, long lasting antibody responses. Interestingly, closely related copolymers have only weak activity, are not adjuvants, or induce inappropriate responses or tolerance. This makes prediction of adjuvant activity complex and imprecise. FNT .sup.4 Hunter, R. L., et al., "The Adjuvant Activity of Nonionic Block Polymer Surfactants I. The Role of Hydrophile-Lipophile Balance", J. Immunol.; Vol. 127, pgs. 1244-1250 (1981) FNT .sup.5 Hunter, R. L., et al., "The Adjuvant Activity of Nonionic Block Polymer Surfactants II. Antibody Formulation and Inflammation Related to the Structure of Triblock and Octablock Copolymers", J. Immunol., Vol. 133, pgs. 3167-3175 (1984) FNT .sup.6 Hunter, R. L., et al., "The Adjuvant Activity of Nonionic Block Polymer Surfactants III. Characterization of Selected Biologically Active Surfaces", Scand. J. Immunol., Vol. 23, pgs. 287-300 (1986)
One might predict that adjuvants whose primary activity was cell stimulation or immunomodulation might work well in combination with the adhesive copolymer adjuvants. The combination of copolymer PLURONIC.RTM. L121 with a threonyl derivative of MDP has been reported to induce better response, particularly a cell mediated immune response, than L121 by itself..sup.7 FNT .sup.7 See U.S. Pat. Nos. 4,606,918 and 4,770,874
Lipopolysaccharides are well-known as B cell mitogens with pronounced effects on macrophages..sup.8 Its adjuvant activities have been know for many years, but its use has been limited by toxicity and variable efficacy. It has been reported in several articles and reviews that the biological activity of the lipopolysaccharides resides in the lipid A portion of the lipopolysaccharide molecule..sup.9 Several strategies have been developed for reducing the toxicity of LPS preparations while maintaining their adjuvant activity. They include the removal of a phosphate group from lipid A to produce monophosphoryl lipid A (MPL) or the removal of one or more fatty acid chains from the lipid A moiety. Some types of LPS, particularly that from Rhodopseudomonas sphaeroides, have an altered lipid A and are inherently non-toxic. FNT .sup.8 Louis, J. A., et al., Lipopolysaccharides: From Immunostimulation to Autoimmunity, Springer Seminars in Immunopathology, Vol. 2, pgs. 215-229 (1979) FNT .sup.9 For example, Galanos, C., et al., "Biological Activities and Immunological Properties of Lipid A", Microbiology, pgs. 269-276 (1977)
The isotype of antibody is very important in resistance to many infections, but little is known about how to produce a particular isotype response. IgG2a has been associated with being a protective isotype for a variety of pathogens, including Trypanosoma cruzi,.sup.10,11 T. musculi.sup.12 and Plasmodium yoelii (malaria) and the bacterium Brucella. IgE antibodies are particularly toxic for parasites in mice. Many parasites including helminths, schistosomes, and nematode larvae naturally stimulate predominantly IgG1 and IgE antibodies. The production of IgG1 and IgE appear to be linked. Each isotype has functional advantages which may be appropriate for neutralizing a particular infectious agent. IgG2a binds most avidly to macrophages, which may influence antibody dependent cell mediated cytotoxicity and phagocytosis and can activate complement. The murine IgG3 isotype is particularly effective in protecting against infections with encapsulated bacteria such as S. pneumoniae. FNT .sup.10 Scott, M. T., et al., "Restricted IgG isotype profiles in T. cruzi infected mice and Chagas' disease patients", Clin. Exp. Immunol., Vol. 58, pgs. 372-379 (1984) FNT .sup.11 Takehara, et al., "Trypanosoma cruzi: role of different antibody classes in protection against infection in the mouse", Exp. Parasitology, Vol. 52, pgs. 137-146 (1981) FNT .sup.12 Wechsler, et al., "Heat labile IgG2a antibodies affect cure of Trypanosoma muculi infection in C57BL/6 mice", J. Immunol. Vol. 137, pgs. 2968-2972 (1986)
Finally, diseases caused by Streptococcus pneumoniae are among the most important bacterial infections of infancy and childhood. A multivalent vaccine containing capsular polysaccharides from 23 types of pneumococci is widely used today. Several studies show that the efficacy of the vaccine in preventing bacteremic illness was 0% in children 2-10 years of age and 49% in persons older than 10 years. There is no convincing evidence that the vaccine is effective for the chronically ill and studies have shown that there is no benefit for the elderly and the institutionalized patients.
By themselves, capsular polysaccharides are thymus independent type 2 (TI-2) antigens which are poorly immunogenic in the very young or very old. TI-2 antigens induce only a restricted number of isotypes, mainly IgM. They induce only a weak memory response, or no memory response, and tolerance is easily induced.
Thus, what is needed in the vaccine art is a composition and method of administering vaccines so that the most efficacious and protective antibody isotype is induced. The vaccine should also be capable of inducing a long-lasting high titer of antibodies.